EGFRvⅢ和CXCR4在人脑胶质母细胞瘤中的表达及其对细胞增殖、侵袭性的影响

发布时间：2018-04-26 17:16

本文选题：表皮生长因子受体Ⅲ型突变体 + 趋化因子受体　；参考：《武汉大学》2014年博士论文

【摘要】：目的：探讨表皮生长因子受体Ⅲ型突变体(epidermal growth factor receptor variant Ⅲ, EGFRvⅢ)和趋化因子受体CXCR4在人脑成胶质母细胞瘤中的表达及其对成胶质母细胞瘤A172细胞增殖、侵袭性迁移的影响和相互关系。方法：应用免疫组织化学染色法检测EGFRvⅢ和趋化因子受体CXCR4在60例人脑成胶质细胞瘤及其瘤旁2cm正常脑组织中的表达水平。建立稳定高表达EGFRvⅢ的成胶质母细胞瘤A172细胞株,应用细胞计数法、MTT法、细胞克隆形成实验以及裸鼠成瘤实验检测EGFRvⅢ对成胶质细胞瘤A172细胞增殖的影响,应用细胞划痕实验和transwell小室侵袭实验检验EGFRvⅢ对成胶质母细胞瘤A172细胞侵袭迁移的作用,再检测高表达EGFRvⅢ后的迁移相关受体蛋白CXCR4的表达情况,进一步探讨EGFRvⅢ促进A172细胞侵袭迁移的可能分子机制,应用si RNA技术抑制CXCR4基因表达后,检测高表达EGFRvⅢ的成胶质母细胞瘤A172细胞的增殖、侵袭迁移能力的变化情况。结果：EGFRvⅢ和CXCR4在成胶质母细胞瘤组织中的阳性表达率明显高于其瘤旁2cm正常脑组织(PEGFRvⅢ0.01; PCXCR40.01)；应用RT-PCR和蛋白印记技术证实EGFRvⅢ高表达的细胞系A172细胞成功建立；与转染空载体对照组比较,高表达EGFRvⅢ的A172细胞增殖能力明显增加；通过连续6天的细胞计数及绘制细胞动态生长曲线发现,细胞培养第5天,A172-EGFRvⅢ细胞数目[(9.1±1.1)×105]约为A172-Control细胞数目[(3.5±0.2)×105]的3倍(P0.05)；对照组和高表达EGFRvIII组的细胞克隆数分别为(50±10)个和(140±25)个,差异有统计学意义(P0.01)；MTT实验检测时,在细胞培养至第3天,稳定高表达EGFRvIII的A172-EGFRvⅢ细胞增殖率约为转染空载体对照组A172-Control细胞的2倍(P0.05)；高表达EGFRvⅢ组的裸鼠皮下移植瘤的体积和质量均高于对照组(P体积0.01；P质量0.01)。在细胞划痕实验中,对照组和高表达EGFRvⅢ组的A172细胞迁移距离分别为(265±75)um和(454±85)um,高表达EGFRvⅢ组约为对照组的1.7倍,差异具有统计学意义(P0.05)。在transwell小室侵袭实验中,对照组和高表达EGFRvⅢ组A172细胞的穿膜数目分别为(8.0±3.1)个/视野和(18.0±2.5)个/视野,差异具有统计学意义(P0.05)。通过RT-PCR和蛋白印记技术发现,高表达EGFRvⅢ可明显促进趋化因子受体CXCR4的表达；进一步的功能实验发现,应用si RNA技术抑制趋化因子受体CXCR4的表达后,绘制细胞动态生长曲线图时,在细胞培养的第5天,A172-EGFRvⅢ-CXCR4siRNA细胞数目[(3.2±1.2)×105]约为A172-EGFRvⅢ-Scrambled siRNA细胞数目[(11.8±0.2)×105]的1/4(P0.05)；细胞克隆形成实验结果,A172-EGFRvⅢ-CXCR4siRNA细胞克隆形成数目约(40±10)个,而A172-EGFRvⅢ-Scrambled siRNA细胞克隆数目高达(98±23)个,二者差异有统计学意义(P0.01); BrdU掺入实验结果显示：A172-EGFRvⅢ-Scrambled siRNA细胞BrdU阳性率为30%, A172-EGFRvⅢ-CXCR4siRNA细胞的BrdU阳性率下降为18%,二者间差异有统计学意义(P0.05)。细胞划痕实验结果：高表达EGFRvIII组的A172细胞迁移距离为(495±75)um, RNA干扰抑制CXCR4表达后细胞迁移距离为(260±67)um,细胞迁移距离降低约50%,差异具有统计学意义(P0.05), transwell小室侵袭实验结果：高表达EGFRvⅢ组A172细胞的穿膜数目为(18.5±2.8)个/视野,RNA干扰抑制CXCR4表达后细胞穿膜数目为(10.1±2.6)个/视野,二组差异具有统计学意义(P0.05)。结论：EGFRvⅢ和CXCR4在人脑成胶质细胞瘤组织中高表达,并且可促进成胶质母细胞瘤A172细胞的增殖、侵袭迁移,通过上调趋化因子受体CXCR4是EGFRvⅢ促进胶质母细胞瘤A172细胞的增值、侵袭性迁移的重要途径之一。
[Abstract]:Objective: To investigate the expression of epidermal growth factor receptor type III mutant (epidermal growth factor receptor variant III, EGFRv III) and chemokine receptor CXCR4 in human glioblastoma and its effect on the proliferation and invasive migration of glioblastoma A172 cells and their relationship. The expression level of EGFRv III and chemokine receptor CXCR4 in 60 human glioblastoma and 2cm normal brain tissue near the tumor was detected by staining. The A172 cell line of glioblastoma stable with high expression of EGFRv III was established. The cell count method, MTT method, cell clone formation, and nude mouse tumorigenesis test were used to detect the formation of EGFRv III. The effect of A172 cell proliferation in stromal tumor cells, the effect of EGFRv III on the invasion and migration of glioblastoma A172 cells and the expression of the migration related receptor protein CXCR4 after the high expression of EGFRv III were detected by the cell scratch test and the Transwell chamber invasion test, and the possibility of EGFRv III to promote the invasion and migration of A172 cells was further explored. Molecular mechanism, using Si RNA technique to suppress CXCR4 gene expression, to detect the proliferation of glioblastoma A172 cells with high expression of EGFRv III and the change of invasion and migration. Results: the positive expression rate of EGFRv III and CXCR4 in glioblastoma tissues was significantly higher than that of the normal brain tissue adjacent to the tumor of 2cm (PEGFRv III 0.01; PCXCR40.0). 1): using RT-PCR and protein imprinting technique, the cell line A172 cells with high expression of EGFRv III were successfully established, and the proliferation ability of A172 cells with high expression of EGFRv III was obviously increased compared with that of the empty vector control group, and the cell culture for fifth days and A172-EGFRv III cells by the continuous 6 days cell count and the plotting of the cell dynamic growth curve. The number [(9.1 + 1.1) * 105]] was about the number of A172-Control cells (3.5 + 0.2) x 105] (P0.05), and the number of cell clones in the control group and the high expression EGFRvIII group were (50 + 10) and (140 + 25) respectively, and the difference was statistically significant (P0.01). The MTT test was used to stabilize the A172-EGFRv III cells expressing EGFRvIII in the cell culture to third days. The proliferation rate was about 2 times (P0.05) of the A172-Control cells in the transfected empty vector control group, and the volume and quality of the subcutaneous transplanted tumor of the high expression EGFRv III group were higher than those of the control group (P volume 0.01, P mass 0.01). The migration distance of the control group and the high expression EGFRv III group was (265 + 75) um and (454 + 85) um respectively in the cell scratch test. The high expression of EGFRv III was about 1.7 times that of the control group, and the difference was statistically significant (P0.05). In the Transwell chamber invasion experiment, the number of A172 cells in the control group and the high expression EGFRv III group A172 cells were (8 + 3.1) / visual field and (18 + 2.5) / visual field, and the difference was statistically significant (P0.05). The results were found by RT-PCR and protein imprinting technique. High expression of EGFRv III can significantly promote the expression of chemokine receptor CXCR4; further functional experiments have found that the number of A172-EGFRv III -CXCR4siRNA cells (3.2 + 1.2) * 105] is about A172-EGFRv III -Scra (3.2 + 1.2) * 105] at the fifth day of cell culture after the application of Si RNA technology to inhibit the expression of chemokine receptor CXCR4. Mbled siRNA cell number [(11.8 + 0.2) x 105] 1/4 (P0.05); cell clone formation experimental results, A172-EGFRv III -CXCR4siRNA cell clone formation number (40 + 10), and A172-EGFRv III -Scrambled siRNA cell clone number is up (98 + 23), two differences have statistical significance (P0.01); BrdU incorporation experiment results show: Dialectical The positive rate of BrdU in -Scrambled siRNA cells was 30%, the positive rate of BrdU in A172-EGFRv III -CXCR4siRNA cells decreased to 18%, and the difference between the two groups was statistically significant (P0.05). The results of the cell scratch test were (495 + 75) um for the high expression of EGFRvIII group, and the cell migration distance of RNA dry disturbance rejection was (260 + 67), and the cell migration distance was (260 + 67). The cell migration distance was reduced by about 50%, the difference was statistically significant (P0.05), and the results of Transwell chamber invasion experiment: the number of A172 cells in the high expression EGFRv III group was (18.5 + 2.8) / visual field, and the number of cell penetrating membrane was (10.1 + 2.6) / visual field after RNA interference inhibition CXCR4 expression. The difference was statistically significant (P0.05). Conclusion: EGFRv III and CXCR4 are highly expressed in human glioblastoma tissue, and can promote the proliferation of glioblastoma A172 cells and invasion and migration. The up regulation of chemokine receptor CXCR4 is one of the important ways to promote the proliferation of A172 cells in glioblastoma and the invasive migration of glioblastoma cells.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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